Developing a Bioanalytical Toolkit to Study the Mechanobiology of Juxtacrine Signaling

开发生物分析工具包来研究近分泌信号传导的力学生物学

• 批准号: 9894683
• 负责人: Khalid S Salaita
• 金额: $ 7.35万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894683
• 关键词: Adaptive Immune System; Amino Acids; Antigens; Area; Atomic Force Microscopy; Autoimmune Diseases; Biochemical Reaction; Biological Assay; CD3 Antigens; Cancerous; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Chemistry; Complex; Coupling; Cytoplasmic Tail; Cytoskeleton; DNA; Data; Enzymes; Fluorescence Polarization; Fluorescence Spectrometry; Goals; Image; Immobilization; Immune response; Immunology; Immunotherapy; Intercellular Junctions; Intuition; Label; Lateral; Ligands; Link; Liquid substance; Literature; Location; Major Histocompatibility Complex; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mechanics; Membrane; Methods; Modeling; Molecular; Molecular Immunology; Molecular Probes; Mutate; Pathway interactions; Peptide Fragments; Peptides; Pharmaceutical Preparations; Receptor Activation; Receptor Signaling; Research Personnel; Resolution; Scanning; Signal Transduction; Signaling Molecule; Signaling Protein; Specificity; Spectrum Analysis; Surface; T cell regulation; T cell therapy; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Testing; Time; Tumor stage; Virus; Work; adaptive immune response; base; biophysical chemistry; cancer cell; chimeric antigen receptor; design; experience; fighting; fluorescence lifetime imaging; high resolution imaging; imaging approach; immune function; immunoengineering; immunological synapse; improved; interdisciplinary approach; interest; mechanical force; mechanotransduction; molecular mechanics; mutant; pathogen; protein complex; public health relevance; ratiometric; receptor; receptor binding; recruit; single molecule; tool; transmission process; vector; virtual

Project Summary/Abstract The long-term goal of this proposal is to better understand how T cells defend against pathogens and eradicate cancerous cells within our bodies. To achieve this goal, T cells continuously crawl seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it initiates activation mechanisms that unleash a potent immune response. Malfunctions in T cell activation are linked with autoimmune disease, while drugs that enhance T cell activation are used to treat cancer. Therefore, there is much interest in understanding the molecular mechanisms of T cell activation. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, there are long standing questions of whether T cells transmit defined forces to their TCR complex and if chemo-mechanical coupling influences immune function. These questions cannot be answered using conventional imaging approaches. The central hypothesis of the proposed work is that advanced mechano- imaging and mechano-analytical approaches will reveal the TCR forces involved in regulation of T cell signaling. Building on our recent breakthroughs in developing high-resolution imaging approaches to map the forces transmitted by cell surface receptors, we will aim to close this gap in our understanding and unravel the mechanical basis of T cell activation. Our preliminary data clearly shows that we have successfully developed the first molecular probes to image the piconewton forces transmitted by the TCR to its ligand during TCR activation. We will test the central hypothesis by first developing molecular force microscopy for the TCR. These probes will test whether the TCR is an anisotropic mechanosensor as proposed in the literature. Next we will map TCR forces within membrane-membrane junctions where the receptor is free to assemble into signaling microclusters. Fluorescence lifetime imaging microscopy (FLIM) and ratiometric probes will be used to map these forces in space and time. Finally, we will use mechanically-triggered enzymes to quantify TCR forces with ultrahigh sensitivity and to tag proximal molecules that are recruited following transmission of TCR forces. The work requires multidisciplinary approaches combining expertise from three investigators that cover the areas of biophysical chemistry, cell biology, and molecular immunology. Importantly, not only will the imaging and quantification techniques developed for this proposal be critical for better understanding the specificity of the adaptive immune system, we expect important implications for the optimal design and implementation of adoptive T cell transfer and chimeric antigen receptors (CARs) in immunotherapy as well as understanding the causes of autoimmune disease.

项目总结/摘要 这项提案的长期目标是更好地了解T细胞如何防御病原体， 根除我们体内的癌细胞为了实现这一目标，T细胞不断爬行， 在其他细胞表面的外源肽片段。一旦T细胞遇到具有外源性的靶细胞， 或突变肽，那么它启动激活机制，释放出一个强大的免疫反应。 T细胞活化功能障碍与自身免疫性疾病有关，而增强T细胞活化的药物 用于治疗癌症。因此，了解T细胞凋亡的分子机制具有重要意义。 细胞激活T细胞活化的第一步涉及T细胞受体（TCR）和T细胞受体（TCR）之间的识别。 由主要组织相容性复合体（pMHC）蛋白呈递的短肽（8-11个氨基酸）。 因为T细胞是高度迁移的，并且当T细胞物理接触抗原时发生抗原识别。 尽管T细胞是靶细胞，但长期以来一直存在这样的问题：T细胞是否将确定的力传递给其TCR复合体 以及化学机械耦合是否影响免疫功能。这些问题不能用 常规成像方法。这项工作的核心假设是，先进的机械- 成像和机械分析方法将揭示参与T细胞调节的TCR力 信号基于我们最近在开发高分辨率成像方法方面的突破， 通过细胞表面受体传递的力，我们的目标是缩小我们理解中的这一差距并解开 T细胞活化的机械基础。我们的初步数据清楚地表明，我们已经成功地开发了 第一种分子探针，用于对TCR在TCR期间传递给其配体的皮牛顿力进行成像， activation.我们将通过首先开发TCR的分子力显微镜来测试中心假设。 这些探针将测试TCR是否是文献中提出的各向异性机械传感器。下 我们将绘制膜-膜连接处的TCR力，在膜-膜连接处，受体可以自由组装成 信号微簇。将使用荧光寿命成像显微镜（FLIM）和比率探针 来绘制这些力在空间和时间上的分布。最后，我们将使用机械触发酶来定量TCR 并标记在TCR传递后募集的近端分子 力.这项工作需要多学科的方法，结合三名调查人员的专业知识， 生物物理化学、细胞生物学和分子免疫学领域。重要的是，不仅 为该提案开发的成像和量化技术对于更好地理解 特异性的适应性免疫系统，我们期待重要的影响，最佳的设计和 过继性T细胞转移和嵌合抗原受体（汽车）在免疫治疗中的实施以及 了解自身免疫性疾病的病因